Therapeutic and prophylactic treatment for colorectal cancer

ABSTRACT

The present invention provides novel methods for treating and preventing colorectal cancer in a subject by administering a composition comprising β-galactosidase, a bacterial culture capable of producing β-galactosidase, such as an S.  thermophilus  culture, or a fraction of the culture comprising β-galactosidase. A kit useful for such methods is also provided. In addition, the present invention provides a composition for treating or preventing colon cancer.

RELATED APPLICATIONS

This application is a divisional of US Application No. 16/750,985 filedJan. 23, 2020, which claims priority to U.S. Provisional Pat.Application No. 62/799,162, filed Jan. 31, 2019, the contents of whichare hereby incorporated by reference in the entirety for all purposes.

BACKGROUND OF THE INVENTION

Colorectal cancer (CRC) is the third most common cancer and the fourthleading cause of cancer death globally, with an estimated incidence of 1million new cases and a mortality of >500 000 deaths per year. Accordingto the data published by the Cancer Registry of the Hong Kong HospitalAuthority, CRC constitutes 16.4% of all new cancer cases and 14.3% ofall cancer deaths in Hong Kong in 2012.

Several intrinsic (e.g., age, male gender, diabetes mellitus, obesityand inflammatory bowel disease) and extrinsic (e.g., cigarette smoking,inadequate intake of fiber, high consumption of alcohol, red meat andhigh-fat diet) factors are associated with increased risks for CRC. Theepidemiology of CRC is under dynamic changes owing to the changingprevalence and distribution of risk factors. In this regard, CRCincidence in many developing countries, including Asian countries, hasincreased 2- to 4-fold over the last two decades and has now reached analarming rate, with Westernization of diet playing a pivotal role.

It has been verified about 100 trillion bacteria exist the in humanintestine and, because of their symbiotic and co-operative relationshipwith the human body, they have a close association with CRC. Theassociation of CRC with altered gut microbiota has been studied indifferent populations, identifying bacteria such as Fusobacteriumnucleatum and Bacteroides fragilis that may be important intumorigenesis. In this regard, F. nucleatum was found to modify thetumor immune microenvironment which in turn promoted colorectalcarcinogenesis. B. fragilis also plays an important oncogenic role byproducing genotoxins to damage DNA in host cells. Prevotella wasreported to be enriched in proximal colon cancer and associated withinterleukin (IL)-17-producing cells. Porphyromonas was also reported tobe associated with CRC in different populations.

Several treatment modalities including surgery, chemotherapy, radiationtherapy and targeted therapy (e.g., cetuximab and bevacizumab) have beendevised to manage CRC. However, the prognosis of patients withmetastatic CRC remains dismal, highlighting the importance of preventionof this disease. The long, stepwise progression of CRC from cellulartransformation to full-blown metastatic lesions has enabled itsprevention through natural compounds or drugs to block or reverse theprocess. In particular, economic analysis suggests that chemopreventioncould be a cost-effective intervention when targeted atintermediate-risk populations following polypectomy. To this end,non-steroidal anti-inflammatory drugs (NSAID_(s)) and cyclooxygenase-2(COX-2) inhibitors have been shown to reduce the occurrence of CRC orits precancerous lesions in high-risk individuals. However, thelong-term use of these agents has been associated with an increased riskof cardiovascular events, posing the concern of high risk benefit ratiofor recommending these agents for CRC chemoprevention. Thechemopreventive effects of other agents including folic acid, calcium,vitamin D, and antioxidants have also been explored but their efficaciesremain to be established. Probiotics are commensal living microorganismsin the human gut. This invention provides a specific probiotic and itssecreted molecules for use in CRC prevention and treatment.

The long, stepwise progression of CRC from cellular transformation tofull-blown metastatic lesions has enabled its prevention through naturalcompounds or drugs to block or reverse the process. However, whentargeted at intermediate-risk populations following polypectomy,chemoprevention could be a cost-effective intervention. It has beenverified that microbiota have a close association with CRC. To this end,the potential use of bacteria specifically depleted in CRC seems anideal approach to prevent CRC development. The present inventorsdiscovered for the first time that S. thermophilus and its secretedmolecules can suppress the growth of CRC in vitro and in vivo. Thisdiscovery thus provides important means for the prevention and treatmentof CRC.

BRIEF SUMMARY OF THE INVENTION

The present inventors have discovered that the bacterium S. thermophilusand its secreted compound(s), such as β-galactosidase, as well as otherbacteria capable of secreting β-galactosidase, can effectively suppressthe proliferation and viability of human colorectal cancer (CRC) cellsand therefore can be used as therapeutic agents for the prevention andtreatment of CRC in both prophylactic and therapeutic applications.

As such, in the first aspect, the present invention provides a methodfor inhibiting cancer cell proliferation. The method includes the stepof contacting cancer cells with a composition comprising an effectiveamount of live S. thermophilus or an extract of a S. thermophilusculture, β-galactosidase or another β-galactosidase-secreting bacteriumor its culture. In some embodiments, the cancer cells are colon cancercells. In some embodiments, the cancer cells, especially colon cancercells, are within a subject’s body. In some embodiments, the subject hasa family history of cancer (such as colon cancer) but has not beendiagnosed with cancer. In other embodiments, the subject has beendiagnosed with cancer, such as colon cancer. In some embodiments, thecomposition used in the claimed method is a culture of S. thermophilusor another β-galactosidase-secreting bacterium, an extract of such abacterial (e.g., S. thermophilus) culture essentially free of thebacteriium (e.g., S. thermophilus), an extract of such a bacterial(e.g., S. thermophilus) culture essentially free of the bacterium (e.g.,S. thermophilus) and retained on a membrane with a molecular weightcut-off (MWCO) of 100 kDa following filtration, or an extract of such abacterial (e.g., S. thermophilus) culture comprising β-galactosidase, orβ-galactosidase, which may be recombinant in nature or may be isolatedfrom a culture of any bacterium capable of secreting β-galactosidase. Insome embodiments, the subject is orally administered a compositioncomprising an effective amount of β-galactosidase, a liveβ-galactosidase-secreting bacterium (such as S. thermophilus), or anextract of a culture of such a bacterium (such as S. thermophilus). Forexample, the composition may be a S. thermophilus culture, an extract ofa S. thermophilus culture essentially free of S. thermophilus, anextract of a S. thermophilus culture essentially free of S. thermophilusand retained on a membrane with a molecular weight cut-off (MWCO) of 100kDa following filtration, or an extract of a S. thermophilus culturecomprising β-galactosidase.

In a second aspect, the present invention provides a kit for treatingcancer or reducing risk of cancer in a subject. The kit includes thesecomponents: (1) a first container containing a first compositioncomprising an effective amount of β-galactosidase, a live culture of abacterium capable of secreting β-galactosidase (such as S.thermophilus), or an extract of such a bacterial (such as S.thermophilus) culture; and (2) a second container containing a secondcomposition comprising an effective amount of an anti-cancer therapeuticagent. In some embodiments, the subject has a family history of cancer,especially colon cancer, but has not been diagnosed with cancer. In someembodiments, the subject has been diagnosed with cancer, especiallycolon cancer. In some embodiments, the first composition is a culture ofa β-galactosidase-secreting bacterium (such as S. thermophilus),especially a live culture, an extract of a β-galactosidase-secretingbacterial (e.g., S. thermophilus) culture essentially free of thebacterium (such as S. thermophilus), an extract of such a bacterial(e.g., S. thermophilus) culture essentially free of the bacterium (e.g.,S. thermophilus) and retained on a membrane with a molecular weightcut-off (MWCO) of 100 kDa following filtration, or an extract of abacterial (e.g., S. thermophilus) culture comprising β-galactosidase, ora composition comprising a β-galactosidase either recombinantly producedor isolated/purified/concentrated from a naturally occurring source. Insome embodiments, the first composition is formulated for oraladministration, for example, the composition may be presented as a fooditem, a beverage, a food supplement, a tablet, a capsule, a paste/cream,a liquid or semi-liquid composition. In some embodiments, thecomposition may be formulated for rectal deposit or insertion.Optionally, the kit further comprises an instruction manual.

In a third aspect, the present invention provides a composition fortreating cancer or reducing risk of cancer comprising (1) an effectiveamount of β-galactosidase, a live culture of a β-galactosidase-secretingbacterium (such as S. thermophilus), or an extract of such a bacterial(e.g., S. thermophilus) culture; and (2) an effective amount of ananti-cancer therapeutic agent; and (3) a physiologically acceptableexcipient. In some embodiments, the cancer is colon cancer. In someembodiments, the composition may be formulated as a rectal suppository.In some embodiments, the extract is an extract of aβ-galactosidase-secreting bacterial (e.g., S. thermophilus) cultureessentially free of the bacterium (e.g., S. thermophilus), an extract ofsuch a bacterial (e.g., S. thermophilus) culture essentially free of thebacterium (e.g., S. thermophilus) and retained on a membrane with amolecular weight cut-off (MWCO) of 100 kDa following filtration, or anextract of a β-galactosidase-secreting bacterial (e.g., S. thermophilus)culture comprising β-galactosidase. In some embodiments, the compositionis formulated for oral administration and is a food item, a beverage, afood supplement, a tablet, a capsule, a paste/cream, a liquid orsemi-liquid composition. In some embodiments, the composition isformulated for rectal suppository.

In a related aspect, the present invention provides use ofβ-galactosidase, a β-galactosidase-secreting bacterium or its liveculture or extract of the culture, such as live S. thermophilus, a S.thermophilus culture, an extract of a S. thermophilus culture asdescribed herein for treating cancer or for reducing cancer risk,especially colon cancer, in subjects that have been diagnosed with thedisease or have known increased risk for the disease although have notyet received a diagnosis of the disease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B shows the prophylactic effect of S. thermophilus onintestinal tumor development in Apc^(Min/+) mice.

FIG. 2 shows the prophylactic effect of S. thermophilus on intestinaltumor development in carcinogen (AOM)-injected mice.

FIG. 3 shows the inhibitory effects of S. thermophilus on the viabilityof cancerous (HCT116, HT29, Caco-2) and normal (NCM460) colonicepithelial cells.

FIG. 4 shows that the killed- S. thermophilus has no effect on CRC celllines.

FIGS. 5A-5B shows the time- and concentration-dependent inhibitoryeffects of St.CM on CRC cell lines.

FIG. 6 shows that St.CM induced apoptotic cell death in cancerous(HCT116, HT29, Caco-2) but not normal (NCM460) colonic epithelial cells.

FIGS. 7A-7D shows the tumor-suppressive effect of >100 KDa fraction fromSt.CM.

FIGS. 8A-8B shows the pro-apoptotic effect of >100 KDa fraction fromSt.CM in CRC cell lines.

FIG. 9 shows the cell cycle-arresting effect of >100 KDa fraction fromSt.CM in CRC cell lines.

FIG. 10 shows the inhibitory effect of >100 KDa fraction from St.CM onthe growth of CRC xenografts in nude mice.

FIG. 11A-11 b shows that >100 KDa fraction from St.CM lost itsinhibitory effects on the viability of CRC cell lines after heatinactivation or co-incubation with proteinase K.

FIGS. 12A-12B shows that the tumor-suppressive fraction separated fromSt.CM contained β-galactosidase.

FIG. 13 shows that the anti-tumor effect of S. thermophilus is mediatedby the secretion of β-galactosidase.

DEFINITIONS

In this disclosure the terms “colorectal cancer (CRC)” and “coloncancer” have the same meaning and refer to a cancer of the largeintestine (colon), the lower part of human digestive system, althoughrectal cancer often more specifically refers to a cancer of the lastseveral inches of the colon, the rectum. A “colorectal cancer cell” is acolon epithelial cell possessing characteristics of colon cancer andencompasses a precancerous cell, which is in the early stages ofconversion to a cancer cell or which is predisposed for conversion to acancer cell. Such cells may exhibit one or more phenotypic traitscharacteristic of the cancerous cells.

In this disclosure the term “or” is generally employed in its senseincluding “and/or” unless the content clearly dictates otherwise.

As used herein, “Streptococcus thermophilus culture” refers to acomposition in which the bacteria S. thermophilus are able toproliferate under the suitable conditions including appropriatetemperature, ventilation, and moisture etc. Typically, a “Streptococcusthermophilus culture” is an aqueous solution comprising the essentialnutrients each in a sufficient quantity for sustaining the growth of S.thermophilus, as well as the bacterium S. thermophilus, and preferablyhas been placed under conditions suitable for S. thermophilusproliferation for a minimal length of time so as to permit S.thermophilus proliferation, e.g., at least 2, 4, 8, 10 and/or up to 12hours, thus containing also compounds secreted by S. thermophilus duringits life cycle. In this application, “an extract” of a “ S. thermophilusculture” refers to any fraction of such a composition or culture havingone or more components of the complete S. thermophilus culture removed,for instance, a bacteria-free fraction has all of the bacteria,especially S. thermophilus, removed (e.g., by centrifugation and/orfiltration through a membrane such as a 0.22 micron membrane) orinactivated (e.g., by heating, chemical treatment, or irradiation). Whenthe presence of S. thermophilus in a composition (such as an extract ofa S. thermophilus culture) yields an essentially the same detectablesignal as a background signal, a composition is referred to as“essentially free of S. thermophilus.” Other possible extracts orfractions may include those having compounds within a certain molecularweight range removed or those having only compounds within a certainmolecular weight range retained (e.g., by filtration through a membraneof a pre-determined molecular weight cut-off or MWCO and recovering theportion that is retained on the membrane or the portion that passesthrough the membrane), or those having its protein components digestedor otherwise inactivated (e.g., by proteolytic, chemical, or heattreatment).

As used herein, “β-galactosidase” refers to a glycoside hydrolase enzymethat catalyzes the hydrolysis of β-galactosides into monosaccharidesthrough the breaking of a glycosidic bond. The term β-galactosidaseincludes variants and homologues originated from different bacterialspecies, such as the β-galactosidase derived from S. thermophilus, whichare identifiable based on similarity in their amino acid sequences andfunctional attributes in their enzymatic activities. For example, a“β-galactosidase” as defined herein shares at least 90%, 95%, or higheramino acid sequence identity with the β-galactosidase derived from S.thermophilus. A “β-galactosidase” may be naturally occurring orrecombinantly produced.

“A β-galactosidase-secreting bacterium” refers to any bacterial speciesthat is capable of producing a detectable level of β-galactosidase,which is released from the bacteria and present in the bacterial cultureand detectable by, e.g., at least one immunoassay such as Western Blot.The bacterium may be a naturally occurring one or may be a recombinantone, i.e., genetically engineered or modified. The production andsecretion of β-galactosidase by such bacterial cells may be constituentor may be inducible.

As used herein, the term “gene expression” is used to refer to thetranscription of a DNA to form an RNA molecule encoding a particularprotein or the translation of a protein encoded by a polynucleotidesequence. In other words, both mRNA level and protein level encoded by agene of interest are encompassed by the term “gene expression level” inthis disclosure.

In this disclosure the term “isolated” nucleic acid molecule means anucleic acid molecule that is separated from other nucleic acidmolecules that are usually associated with the isolated nucleic acidmolecule. Thus, an “isolated” nucleic acid molecule includes, withoutlimitation, a nucleic acid molecule that is free of nucleotide sequencesthat naturally flank one or both ends of the nucleic acid in the genomeof the organism from which the isolated nucleic acid is derived (e.g., acDNA or genomic DNA fragment produced by PCR or restriction endonucleasedigestion). Such an isolated nucleic acid molecule is generallyintroduced into a vector (e.g., a cloning vector or an expressionvector) for convenience of manipulation or to generate a fusion nucleicacid molecule. In addition, an isolated nucleic acid molecule caninclude an engineered nucleic acid molecule such as a recombinant or asynthetic nucleic acid molecule. A nucleic acid molecule existing amonghundreds to millions of other nucleic acid molecules within, forexample, a nucleic acid library (e.g., a cDNA or genomic library) or agel (e.g., agarose, or polyacrylamine) containing restriction-digestedgenomic DNA, is not an “isolated” nucleic acid.

The term “nucleic acid” or “polynucleotide” refers to deoxyribonucleicacids (DNA) or ribonucleic acids (RNA) and polymers thereof in eithersingle- or double-stranded form. Unless specifically limited, the termencompasses nucleic acids containing known analogs of naturalnucleotides that have similar binding properties as the referencenucleic acid and are metabolized in a manner similar to naturallyoccurring nucleotides. Unless otherwise indicated, a particular nucleicacid sequence also implicitly encompasses conservatively modifiedvariants thereof (e.g., degenerate codon substitutions), alleles,orthologs, single nucleotide polymorphisms (SNPs), and complementarysequences as well as the sequence explicitly indicated. Specifically,degenerate codon substitutions may be achieved by generating sequencesin which the third position of one or more selected (or all) codons issubstituted with mixed-base and/or deoxyinosine residues (Batzer et al.,Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem.260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98(1994)). The term nucleic acid is used interchangeably with gene, cDNA,and mRNA encoded by a gene.

The term “gene” means the segment of DNA involved in producing apolypeptide chain; it includes regions preceding and following thecoding region (leader and trailer) involved in thetranscription/translation of the gene product and the regulation of thetranscription/translation, as well as intervening sequences (introns)between individual coding segments (exons).

In this application, the terms “polypeptide,” “peptide,” and “protein”are used interchangeably herein to refer to a polymer of amino acidresidues. The terms apply to amino acid polymers in which one or moreamino acid residue is an artificial chemical mimetic of a correspondingnaturally occurring amino acid, as well as to naturally occurring aminoacid polymers and non-naturally occurring amino acid polymers. As usedherein, the terms encompass amino acid chains of any length, includingfull-length proteins (e.g., β-galactosidase derived from S.thermophilus), wherein the amino acid residues are linked by covalentpeptide bonds.

The term “amino acid” refers to refers to naturally occurring andsynthetic amino acids, as well as amino acid analogs and amino acidmimetics that function in a manner similar to the naturally occurringamino acids. Naturally occurring amino acids are those encoded by thegenetic code, as well as those amino acids that are later modified,e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. For thepurposes of this application, amino acid analogs refers to compoundsthat have the same basic chemical structure as a naturally occurringamino acid, i.e., an a carbon that is bound to a hydrogen, a carboxylgroup, an amino group, and an R group, e.g., homoserine, norleucine,methionine sulfoxide, methionine methyl sulfonium. Such analogs havemodified R groups (e.g., norleucine) or modified peptide backbones, butretain the same basic chemical structure as a naturally occurring aminoacid. For the purposes of this application, amino acid mimetics refersto chemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid.

Amino acids may include those having non-naturally occurringD-chirality, as disclosed in WO01/12654, which may improve the stability(e.g., half-life), bioavailability, and other characteristics of apolypeptide comprising one or more of such D-amino acids. In some cases,one or more, and potentially all of the amino acids of a therapeuticpolypeptide have D-chirality.

Amino acids may be referred to herein by either the commonly known threeletter symbols or by the one-letter symbols recommended by the IUPAC-IUBBiochemical Nomenclature Commission. Nucleotides, likewise, may bereferred to by their commonly accepted single-letter codes.

As used in this application, an “increase” or a “decrease” refers to adetectable positive or negative change in quantity from a comparisoncontrol, e.g., an established standard control. An increase is apositive change that is typically at least 10%, or at least 20%, or 50%,or 100%, and can be as high as at least 2-fold or at least 5-fold oreven 10-fold of the control value. Similarly, a decrease is a negativechange that is typically at least 10%, or at least 20%, 30%, or 50%, oreven as high as at least 80% or 90% of the control value. Other termsindicating quantitative changes or differences from a comparative basis,such as “more,” “less,” “higher,” and “lower,” are used in thisapplication in the same fashion as described above. In contrast, theterm “substantially the same” or “substantially lack of change”indicates little to no change in quantity from the standard controlvalue, typically within ± 10% of the standard control, or within ± 5%,2%, 1%, or even less variation from the standard control.

The term “inhibiting” or “inhibition,” as used herein, refers to anydetectable negative effect on a target biological process, such as RNAtranscription, protein expression, cell proliferation, cellular signaltransduction, cell proliferation, tumorigenicity, metastatic potential,and recurrence of a disease/condition. Typically, an inhibition isreflected in a decrease of at least 10%, 20%, 30%, 40%, or 50% in thetarget process (e.g., CRC cell proliferation rate) upon application ofan inhibitor, when compared to a control where the inhibitor is notapplied.

The term “treat” or “treating,” as used in this application, describesto an act that leads to the elimination, reduction, alleviation,reversal, or prevention or delay of onset or recurrence of any symptomof a relevant condition. In other words, “treating” a conditionencompasses both therapeutic and prophylactic intervention against thecondition.

The term “effective amount” as used herein refers to an amount of agiven substance that is sufficient in quantity to produce a desiredeffect. For example, an effective amount of S. thermophilus culture, ora fraction thereof, especially a fraction comprising the β-galactosidasederived from S. thermophilus, is the amount of said culture or fractionthereof to achieve a decreased level of a target process, e.g., CRCcellular proliferation or viability, such that the risk, symptoms,severity, and/or recurrence change of colon cancer are reduced,reversed, eliminated, prevented, or delayed of the onset in a patientwho has been given the S. thermophilus culture or a fraction thereof fortherapeutic and/or prophylactic purposes. An amount adequate toaccomplish this is defined as the “therapeutically effective dose.” Thedosing range varies with the nature of the therapeutic agent beingadministered and other factors such as the route of administration andthe severity of a patient’s condition.

The term “subject” or “subject in need of treatment,” as used herein,includes individuals who seek medical attention due to risk of, oractual suffering from, colon cancer. Subjects also include individualscurrently undergoing therapy that seek manipulation of the therapeuticregimen. Subjects or individuals in need of treatment include those thatdemonstrate symptoms of colon cancer or are at risk of suffering fromcolon cancer or its symptoms. For example, a subject in need oftreatment includes individuals with a genetic predisposition or riskfactors including family history for colon cancer or personal medicalhistory and/or life style choices such as those described in thisdisclosure that tend to bring about increased risk of the disease, thosethat have suffered relevant symptoms in the past, those that have beenexposed to a triggering substance or event, as well as those sufferingfrom chronic or acute symptoms of the condition. A “subject in need oftreatment” may be at any age of life.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

Colorectal cancer patients often face a grim prognosis when the diseaseis detected in its later stages. Early detection and treatment,including prophylactic treatment, of colorectal cancer is thereforecritical for improving patient clinical outcome.

The present inventors discovered for the first time that aβ-galactosidase-secreting bacterium (such as S. thermophilus) and itssecreted compound or compounds (especially β-galactosidase) can providesignificant benefits in preventing and treating colorectal cancer (CRC).More specifically, it is revealed by the inventors that the probiotic S.thermophilus and its conditioned-medium (St.CM) containingβ-galactosidase can inhibit CRC cell proliferation in vitro and in vivo.Further, it can also induce CRC cell apoptosis and cell cycle arrest.Oral gavage of S. thermophilus to Apc^(min/+) mice and AOM induced CRCmice can slow down the tumor growth and reduce the tumor sizesignificantly. On the other hand, loss of β-galactosidase secretioncapability in bacteria leads to abolition of the anti-cancer activities.Overall, this study shows that probiotic bacteria capable of producingβ-galactosidase and secreted molecule(s), especially β-galactosidase,can serve as a novel therapeutic agent for treating CRC in bothprophylactic and therapeutic applications.

II. Treatment of Colon Cancer

By illustrating the inhibitory effect of β-galactosidase-secretingbacteria such as S. thermophilus and their secreted compound(s), e.g.,β-galactosidase, on cancer cells such as colon cancer cells, the presentinvention provides a novel method for treating patients who have beendiagnosed with cancer, e.g., colon cancer, as well as subjects who havenot been diagnosed with the disease but are at increased risk ofdeveloping the cancer, for example, due to genetic predisposition,family history of cancer (especially colon cancer), and/or personaltraits and medical background such as age (50 years or older), gender(male), diabetes mellitus, obesity, and inflammatory bowel disease, aswell as smoking and certain dietary choices (e.g., inadequate intake offiber, high consumption of alcohol, red meat, and high salt/high fatfoods). In accordance with this disclosure, a β-galactosidase-producingbacterial culture, such as S. thermophilus culture, or an extract orfraction thereof containing the bacterium and/or its secretedcompound(s) (such as β-galactosidase), especially an extract thatcontains molecules secreted by the bacteria cells with a molecularweight greater than 100 kDa and especially contains β-galactosidase, canbe used for administration to such individuals for the purpose toprevent or reduce the risk of the rise of colon cancer among at-riskindividuals in prophylactic applications as well as to treat coloncancer patients in therapeutic applications for the purpose to suppresscolon cancer cell growth and metastasis, possibly in conjunction withother conventional therapy regiments such as surgical intervention,chemotherapy, radiotherapy, immunotherapy or any combinations thereof.

A. Bacterial Culture and Extracts

A culture of a β-galactosidase-secreting bacterium (which may be anaturally-occurring bacterial species such as S. thermophilus or arecombinant bacterial cell line generated by artificially introducingone or more exogenous genes encoding the enzyme or enhancing theexpression of endogenous β-galactosidase) can be established accordingto methods known in the art and described herein under appropriateconditions. Typically, a live β-galactosidase-secreting bacterial (suchas S. thermophilus) culture can be obtained by culturing the bacteriumin a liquid medium containing essential nutrients under propertemperature for 12-24 hours so as to permit the bacterial cells toproliferate in the medium. The live bacterial (e.g., S. thermophilus)culture may be directly used for producing a composition in a suitableformula or form for administration to a subject in need thereof. In thealternative, an extract or fraction of such a live culture of aβ-galactosidase-secreting bacterium (such as S. thermophilus) may beused instead of the full culture for formulation. For this purpose, abacteria-free culture extract may be generated from the live culture,e.g., after processing the full culture by centrifugation and/orfiltration to remove essentially all of the main bacterial cells (suchas S. thermophilus) and other bacteria potentially present from theculture. Also, an extract of the β-galactosidase-secreting bacterialculture (such as an S. thermophilus culture) that is essentially free ofthe bacterial cells such as S. thermophilus or other bacteria butcontains all compounds secreted by the bacterial cells such as S.thermophilus within a preferred molecular weight range may be obtainedby a filtration process utilizing a suitable membrane of a desirablepore size. For example, a membrane having a molecular weight cut-off of100 kDa can be used in filtration, which leaves compounds over themolecular weight retained on the membrane (the retentate portion) whileallowing compounds under the molecular weight passing through themembrane (the filtrate portion). Other possible extracts or fractions ofa live β-galactosidase-secreting bacterial culture, such as a S.thermophilus culture, may include those that has one particular type ofmolecules (such as protein) all removed or enriched. For example, oneextract may have highly enriched β-galactosidase secreted by aβ-galactosidase-secreting bacterium such as S. thermophilus in itsculture.

B. Β-Galactosidase

As shown by the present inventors, β-galactosidase is the compoundessential for the anti-cancer activity a β-galactosidase-secretingbacterium is able to support. Such, one aspect of the present inventionis the use of β-galactosidase for therapeutic and prophylactic purposes.

The protein β-galactosidase can be obtained via a variety of means,including but not limited to, recombinant production of β-galactosidaseby engineered prokaryotic or eukaryotic cells, isolation/purification orpartial isolation/enrichment of a naturally-occurring proteinβ-galactosidase from a source such as a β-galactosidase-secretingbacterial culture. Thus, not only a naturally-occurring β-galactosidasefrom any bacterial culture, including its variants or homologs with thesame type of enzymatic activity-as a glycoside hydrolase enzyme capableof catalyzing the hydrolysis of β-galactosides into monosaccharides bybreaking the glycosidic bond, can be used in the claimed methods andcompositions of the present invention, any one of the recombinant formsof β-galactosidase protein can also be used. This includes recombinantβ-galactosidase proteins of different forms, which may comprise sequencemodifications such as deletions, insertions, or substitutions of one ormore amino acid residues or may encompass one or more heterologouspeptide sequences (one from a source different from the origin of theβ-galactosidase) such as affinity or epitope tags, but whichnevertheless retain the same type of enzymatic activity of catalyzingthe hydrolysis of β-galactosides.

C. Pharmaceutical Compositions 1. Formulations

β-galactosidase, a live bacterial culture capable of producingβ-galactosidase (such as a S. thermophilus culture), or an extractthereof, especially one containing β-galactosidase, is useful in themanufacture of a pharmaceutical composition or a medicament. Apharmaceutical composition or medicament can be administered to asubject for the treatment of colon cancer, especially for prophylaxis.

β-galactosidase, a β-galactosidase-producing bacterial culture such as aS. thermophilus culture, or its extract used in the treatment method ofthe present invention are useful in the manufacture of a pharmaceuticalcomposition or a medicament or a food item including a beverage or foodsupplement in conjunction or mixture with one or more physiologicallyacceptable excipients or carriers suitable for administration.

An exemplary pharmaceutical composition for such therapeutic usecomprises (i) an effective amount of β-galactosidase, a live culture ofa β-galactosidase-secreting bacterium, such as S. thermophilus culture,or an extract as described herein, and (ii) a pharmaceuticallyacceptable excipient or carrier. The terms pharmaceutically-acceptableand physiologically-acceptable are used synonymously herein.

Pharmaceutical compositions or medicaments for use in the presentinvention can be formulated by standard techniques using one or morephysiologically acceptable carriers or excipients. Suitablepharmaceutical carriers are described herein and in “Remington’sPharmaceutical Sciences” by E.W. Martin. Compounds and agents of thepresent invention and their physiologically acceptable salts andsolvates can be formulated for administration by any suitable route,including via inhalation, topically, nasally, orally, parenterally, orrectally, depending on the anatomic sites for delivery, such as wherethe cancer is present or likely to arise: for example, colon for coloncancer, stomach for gastric cancer, and skin for skin cancer.

Typical formulations for topical administration include creams,ointments, sprays, lotions, and patches. The pharmaceutical compositioncan, however, be formulated for any type of administration, e.g.,intradermal, subdermal, intravenous, intramuscular, intranasal,intracerebral, intratracheal, intraarterial, intraperitoneal,intravesical, intrapleural, intracoronary or intratumoral injection,with a syringe or other devices. Formulation for administration byinhalation (e.g., aerosol), or for oral, rectal, or vaginaladministration is also contemplated.

2. Routes of Administration

Suitable formulations for topical application, e.g., to the skin andeyes, are preferably aqueous solutions, ointments, creams or gelswell-known in the art. Such may contain solubilizers, stabilizers,tonicity enhancing agents, buffers and preservatives.

Suitable formulations for transdermal application include an effectiveamount of a composition of the present invention with one or morecarriers. Preferred carriers include absorbable pharmacologicallyacceptable solvents to assist passage through the skin of the host. Forexample, transdermal devices are in the form of a bandage comprising abacking member, a reservoir containing the compound optionally withcarriers, optionally a rate controlling barrier to deliver the compoundto the skin of the host at a controlled and predetermined rate over aprolonged period of time, and means to secure the device to the skin.Matrix transdermal formulations may also be used.

For oral administration, a pharmaceutical composition or a medicament ora food item or a food supplement can take the form of, for example, atablet or a capsule prepared by conventional means with apharmaceutically acceptable excipient. Preferred are tablets and gelatincapsules comprising the active ingredient, i.e., β-galactosidase, aβ-galactosidase-secreting bacterial culture such as a S. thermophilusculture, or an extract thereof especially containing β-galactosidase,together with (a) diluents or fillers, e.g., lactose, dextrose, sucrose,mannitol, sorbitol, cellulose (e.g., ethyl cellulose, microcrystallinecellulose), glycine, pectin, polyacrylates and/or calcium hydrogenphosphate, calcium sulfate, (b) lubricants, e.g., silica, talcum,stearic acid, its magnesium or calcium salt, metallic stearates,colloidal silicon dioxide, hydrogenated vegetable oil, corn starch,sodium benzoate, sodium acetate and/or polyethyleneglycol; for tabletsalso (c) binders, e.g., magnesium aluminum silicate, starch paste,gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose,polyvinylpyrrolidone and/or hydroxypropyl methylcellulose; if desired(d) disintegrants, e.g., starches (e.g., potato starch or sodiumstarch), glycolate, agar, alginic acid or its sodium salt, oreffervescent mixtures; (e) wetting agents, e.g., sodium lauryl sulphate,and/or (f) absorbents, colorants, flavors and sweeteners.

Tablets may be either film coated or enteric coated according to methodsknown in the art. Liquid preparations for oral administration can takethe form of, for example, solutions, syrups, or suspensions, or they canbe presented as a dry product for constitution with water or othersuitable vehicle before use. Such liquid preparations can be prepared byconventional means with pharmaceutically acceptable additives, forexample, suspending agents, for example, sorbitol syrup, cellulosederivatives, or hydrogenated edible fats; emulsifying agents, forexample, lecithin or acacia; non-aqueous vehicles, for example, almondoil, oily esters, ethyl alcohol, or fractionated vegetable oils; andpreservatives, for example, methyl or propyl-p-hydroxybenzoates orsorbic acid. The preparations can also contain buffer salts, flavoring,coloring, and/or sweetening agents as appropriate. If desired,preparations for oral administration can be suitably formulated to givecontrolled release of the active compound. For use in food items, thecomposition or active ingredient (e.g., β-galactosidase, bacterial cellscapable of producing β-galactosidase, a live culture thereof, or anextract thereof containing β-galactosidase) of this invention may bedirectly added to conventional food items such as a supplement or may bemixed into a beverage or snack.

Active compounds and agents of the present invention can be formulatedfor parenteral administration by injection, for example by bolusinjection or continuous infusion. Formulations for injection can bepresented in unit dosage form, for example, in ampoules or in multi-dosecontainers, with an added preservative. Injectable compositions arepreferably aqueous isotonic solutions or suspensions, and suppositoriesare preferably prepared from fatty emulsions or suspensions. Thecompositions may be sterilized and/or contain adjuvants, such aspreserving, stabilizing, wetting or emulsifying agents, solutionpromoters, salts for regulating the osmotic pressure and/or buffers.Alternatively, the active ingredient (e.g., β-galactosidase, bacterialcells capable of producing β-galactosidase, a live culture thereof, oran extract thereof containing β-galactosidase) can be in powder form forconstitution with a suitable vehicle, for example, sterile pyrogen-freewater, before use. In addition, they may also contain othertherapeutically valuable substances. The compositions are preparedaccording to conventional mixing, granulating or coating methods,respectively, and contain about 0.1 to 75%, preferably about 1 to 50%,or about 5 to 25%, of the composition or active ingredients derived froma β-galactosidase-producing bacterial culture such as S. thermophilusculture, e.g., β-galactosidase.

For administration by inhalation, the composition or active ingredientsderived from the β-galactosidase-producing bacterial culture such as anS. thermophilus culture (e.g., β-galactosidase) may be convenientlydelivered in the form of an aerosol spray presentation from pressurizedpacks or a nebulizer, with the use of a suitable propellant, forexample, dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. In thecase of a pressurized aerosol, the dosage unit can be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof, for example, gelatin for use in an inhaler or insufflator can beformulated containing a powder mix of the compound and a suitable powderbase, for example, lactose or starch.

The compounds or active ingredients of this invention can also beformulated in rectal compositions, for example, suppositories orretention enemas, for example, containing conventional suppositorybases, for example, cocoa butter or other glycerides.

Furthermore, the compounds or active ingredients can be formulated as adepot preparation. Such long-acting formulations can be administered byimplantation (for example, subcutaneously or intramuscularly) or byintramuscular injection. Thus, for example, the compounds or activeingredients can be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

In some cases, a pharmaceutical composition or medicament of the presentinvention comprises (i) an effective amount of a composition asdescribed herein that suppresses the proliferation or promotes the deathof cancer cells especially colon cancer cells, and (ii) anothertherapeutic agent especially a known anti-cancer drug such as achemotherapeutic agent against cancer including colon cancer. When usedwith a composition or active ingredient of the present invention, suchtherapeutic agent may be used individually, sequentially, or incombination with one or more other such therapeutic agents (e.g., afirst therapeutic agent, a second therapeutic agent, and a compositionof the present invention). Administration may be by the same ordifferent route of administration or together in the same pharmaceuticalformulation.

III. Compositions and Kits

The invention provides compositions and kits for practicing the methodsdescribed herein to treat cancer or to reduce the risk of cancerdevelopment, especially colon cancer, in a subject. For example, acomposition useful for the therapeutic or prophylactic applicationstypically contains (1) an effective amount of β-galactosidase, a liveβ-galactosidase-secreting bacterium such as S. thermophilus, aβ-galactosidase-secreting bacterial culture such as a S. thermophilusculture, or an extract of such culture, especially containingβ-galactosidase; and (2) a carrier or excipient, especially one that isappropriate for formulation suitable for oral administration. Forexample, one extract of such a bacterial culture such as an S.thermophilus culture is an extract that is essentially free of thebacterial cells (e.g., S. thermophilus and/or other bacterial cells) butretains compounds secreted by the β-galactosidase-secreting bacterium(e.g., S. thermophilus) with a molecular weight greater than 100 kDa,including β-galactosidase. In some cases the composition will beformulated for oral administration or rectal suppository. Optionally thecomposition will further comprise a second therapeutically effectiveagent, such as a chemotherapeutic agent known to be effective for cancertreatment.

Kits for carrying out the treatment methods of the present inventiontypically include (1) a first container containing a composition of thisinvention, e.g., one comprising an effective amount of β-galactosidase,a live β-galactosidase-secreting bacterium such as S. thermophilus, aβ-galactosidase-secreting bacterial culture such as S. thermophilusculture, or an extract of such culture, for example, an extract that isessentially free of bacteria (e.g., S. thermophilus and/or otherbacterial cells) but retains compounds secreted by S. thermophilus witha molecular weight greater than 100 kDa, including β-galactosidase; and(2) a second container containing an effective amount of a secondtherapeutically effective agent, such as a chemotherapeutic agent knownto be effective for cancer treatment. Kits may further include aninstruction manual to guide users for properly dispensing thecompositions.

EXAMPLES

The following examples are provided by way of illustration only and notby way of limitation. Those of skill in the art will readily recognize avariety of non-critical parameters that could be changed or modified toyield essentially the same or similar results.

Example 1: S. Thermophilus and Its Inhibitory Effect on CRC Introduction

The present inventors have identified S. thermophilus and its secretedmolecules as novel prophylactics or therapeutics for the prevention ortreatment of CRC. More specifically, the inventors show that,coculturing S. thermophilus with colon cells can inhibit cancer cellproliferation in vitro and in vivo. Compounds secreted by the bacteriacan also have the same effect in vitro and in vivo. Moreover, thesecreted molecules can induce programmed cell death and cause cell cyclearrest at G0/G1 phase. The administration of this specific probioticscan prevent CRC formation in animals. Therefore, S. thermophilus and itssecreted molecules are useful for the prevention and treatment of CRC.

Materials and Methods Bacterial Strain and Culture Condition

Streptococcus thermophilus [ATCC 19258] was purchased from the AmericanTissue Culture Collection (Manassas, VA) and cultured in Brain HeartInfusion (BHI) Broth (CM1135B; Thermo Fisher Scientific, West PalmBeach, FL) for 24 hours at 37° C. in aerobic condition. A nonpathogenichuman commensal intestinal bacteria, Escherichia coli strain MG1655 wasused as a control and was cultured in the same condition as S.thermophilus. The mutant S. thermophilus (LacZ knockout) was constructedby homologous recombination and were cultured in selective antibiotic(50 ug/ml kanamycin) BHI broth.

Cell Culture

CRC cell lines HCT116, HT29 and Caco-2 were obtained from the AmericanType Culture Collection. Human normal colon epithelial cell line NCM460was obtained from INCELL Corporation (San Antonio, TX). All the celllines were growth in high-glucose Dulbecco’s Modified Eagle’s Medium(DMEM) (Gibco BRL, Grand Island, NY) supplemented with 10% (vol/vol)fetal bovine serum (FBS), 1% penicillin/streptomycin in a humidifiedatmosphere containing 5% CO₂.

Preparation of the Conditioned Medium

When the density of bacteria reached at OD=0.5 at A600nm, the bacteriaculture medium will be centrifuged (1,000×g for 15 min) and filteredthrough a 0.2-µm pore-size filter to obtain the conditioned-medium.

The Isolation of the Anti-tumor Molecule

The prepared conditioned-medium was separated with a molecular weightcutoff spin column (Merck KGaA, Darmstadt, Germany). The >100 KDafraction was obtained by centrifuge the conditioned-medium through a100,000 NMWL membrane.

Characterization of the Anti-Tumor Molecules

The anti-tumor molecules were digested by protease K at 55° C. for 2 hor were heat-inactivated at 100° C. for 30 min. The protease K was theninactivated at 95° C. for 10 min.

Silver Stain for Mass Spectrometry

The anti-tumor molecule was separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (5%) (SDS-PAGE). The silver stain wasperformed following the instructions provided by the manufacture (ThermoScientific, Rockford, USA). After staining, the >100 KDa fraction bandswere removed and in-gel digestion was performed. The obtainedsupernatant was subjected to MS analysis.

Mass Spectrometry

The fraction was diluted in methanol and injected into a Nano FrontiereID Liquid Chromatography Mass Spectrometer (Hitachi High-technologies,Japan) at 3µl/min. The detected signal was analyzed according theexisted database.

Colony Formation Assay

Colon cells (1000 per well) were seeded on six-well plates, followingthe treatment of St.CM (1% St.CM in DMEM). Brain heart infusion (BHI)and the conditioned-medium of E. coli (Ec.CM) were used as control. Thetreatment medium was changed every 3 days. After culturing for 14-18days, cells were fixed with 70% ethanol and stained with 0.5% crystalviolet solution. Colony with more than 50 cells was counted. Allexperiments were performed three times in triplicate.

Cell Viability Assay

Cell viability was determined by the3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay. Foreach well in the 96-well plate, 1,000 cells were seeded and treated with1% vol/vol of bacteria conditioned medium or bacteria directly. After24, 48 and 72 hr, 10 µL MTT (5 mg/ml) reagent was added into the culturemedium, respectively. The cells were then incubated at 37° C. until theintracellular purple formazan crystals are visible, which were thendissolved by DMSO. Absorbance of the samples was measured at 570 nm.

Flow Cytometry

For cell cycle analysis, the treated cells were fixed by 70% ethanolovernight. Cells were then stained with 50 µg/mL propidium iodide (PI)(BD Pharmingen, San Jose, CA, USA) for 30 minutes at 4° C. in dark.10,000 cells were counted by FACSAria cell sorter (BD Biosciences,Franklin Lakes, NJ, USA) and cell-cycle profiles were analyzed by ModFit3.0 software (Verity Software House, Topsham, ME, USA). The proportionof apoptotic cells was evaluated using Annexin V apoptosis assay. Thetreated cells were collected and resuspended in 100 µL, annexin-bindingbuffer (10 mM HEPES, 140 mM NaCl and 2.5 mM CaCl₂, pH 7.4) containing 5µL Annexin V conjugated with Allophycocyanin and 50 µg/mL PI (BDPharmingen). After incubation for 15 min at room temperature, cells weremixed with additional 400 µL of ice-cold annexin-binding buffer andanalyzed using FACSAria cell sorter (BD Biosciences).

Ki-67 Staining

Cell proliferation was assayed by immunoperoxidase staining withanti-Ki-67 antibody (ab833; Abcam,Cambridge, UK). Negative controls wererun by replacing the primary antibody with nonimmune serum. Theproliferation index was determined by counting the numbers of positivestaining cells as percentages of the total number of tumor cells. Atleast 1000 tumor cells were counted each time.

Western Blotting Analysis

Total protein was isolated and separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (6%-12%) (SDS-PAGE). The protein inSDS-PAGE were then transferred onto polyvinylidene difluoride (PVDF)membranes (EMD Millipore, Billerica, MA, USA) for about 1-2 hours, whichwas then blocked with 10% non-fat milk in 0.05% Tris-based saline-Tween20 for 2 hours at RT. The membrane was incubated with primary antibodiesovernight at 4° C. and then with secondary antibody at RT for 1 hour.The protein band intensities were detected by ECL Plus Western blottingDetection Reagents (GE Healthcare).

Histopathology

At the end of animal experiment, the fresh tissues were flattened onfilter paper and fixed overnight in 10% buffered formalin forhistopathological assessment. Sections of 5 µm were stained with H&E forhistologic diagnosis by an experienced pathologist. Dysplasia wasdetermined following the latest World Health Organization’sClassification of TuMORS OF THE Digestive System.

In Vivo Tumorigenicity - Xenograft Tumor Model

HCT116 (1× 106 cells in 0.1 ml phosphate-buffered saline) were injectedsubcutaneously into the right dorsal flank of four 4-week-old maleBalb/c nude mice, separately (6/group). Intro-tumoral injection of theconditioned-medium was performed 3 times/week. Tumor diameter wasmeasured every 2 days. Tumor volume (mm³) was estimated by measuring thelongest and shortest diameter of the tumor and calculating as follows:volume = (shortest diameter)2 x (longest diameter) × 0.5. After 19 days,the mice were sacrificed, and the tumors were weighed and fixed informalin for histological analysis. All experimental procedures wereapproved by the Animal Ethics Committee of the Chinese University ofHong Kong.

In Vivo Tumorigenicity - Colitis-Associated Cancer Model

6-week-old male conventional C57BL/6 wild-type mice were subjected 6consecutive injection of AOM (10 mg/kg, intraperitoneal injection) at1-week intervals, followed by the presence or absence of an antibioticcocktail (0.1 g/L of Vancomycin, 0.1 g/L of Neomycin, 0.1 g/L ofPenicillin and 0.2 g/L of Metronidazole) in drinking water for 2 weeks.The mice were then gavaged with 1 × 108 CFU of S.termophilus, E.coliMG1655 or the same volume of PBS every day for 20 weeks for thedevelopment of neoplastic lesions. Sulindac (180 ppm/ week) was used asa positive control.

In Vivo Tumorigenicity - APC^(Min/+) model

C57BL/6J-ApcMin/J mice, which harbor germline mutation in the tumorsuppressor gene APC and develop intestinal polyps spontaneously, havebeen purchased from the Jackson Laboratory (Bar Harbor, ME, USA) and arecurrently maintained in the animal facility at our University.Genotyping will be conducted by routine allele-specific PCR assay. Thesame treatment with AOM model was used in eight-week-old maleAPC^(min/+) mice and they were raised till 20 weeks for the evaluationof the bacteria treatment efficacy.

Statistical Analysis

Data are expressed as mean ± standard deviation (SD) from 3 dependentexperiments. The independent Student t test was used to compare thedifference between two groups. One-way analysis of variance (ANOVA) wasused to compare the difference between multiple groups. Differences withP-value<0.05 were considered statistically significant. All tests wereperformed using GraphPad Prism, version 6.0.

S. Thermophilus Mutant Strain Construction

The mutant S. thermophilus (LacZ knockout) was constructed by homologousrecombination. Kanamycin resistance gene fragment was amplified from thepCR2.1-TOPO plasmid. Sequences around 1 kb upstream and downstream with500 bp space were amplified from the genomic DNA of S. thermophilus,respectively. Subsequently, three DNA fragments, including the sequencesaround 1kb upstream of the LacZ gene encoding β-galactosidase and 900 bpkanamycin resistance gene, and sequences around 1 kb downstream of theLacZ gene, were cleaved by restriction enzymes and then linkedsuccessively. The recombinant DNA fragment was linked into thepCR2.1-TOPO plasmid using the T-A cloning technique. The recombinantpCR2.1-TOPO plasmid was transformed into the competent cells of S.thermophilus by electroporation (1.8 kV, 200 Ω, and 25 µF). Thecompetent cell was constructed by exposing S. thermophilus to 50 ml BHIbroth containing 20 mM D-L threonine for 2-2.5 hours to reach an opticaldensity (OD)600 of 0.2-0.3. The cells pellets (4,500×g, 15 minutes) werewashed with 5ml electroporation buffer (7 mM HEPES, 1 mM MgCl₂, pH 6.0)and resuspended to an OD600 of 20 in the same buffer to obtain theelectrocompetent cells. The electroporated cells were spread ontopre-warmed BHI agar plates containing X-gal and the representativeputative transformant colonies were then cultured in selectiveantibiotic (50 ug/ml kanamycin) for genomic isolation andβ-galactosidase activity test. The presence of the relevant plasmidspecies was confirmed by agarose gel electrophoretic examination and thederivatives were ensured by 16S ribosomal RNA gene sequencing.

Results Streptococcus Thermophilus Reduces Colonic Tumorigenesis in ApcMin/+ Mice With or Without Microbiota Depletion

To evaluate whether S.thermophilus could have anti-tumorigenic effect inmice. 6 weeks old Non-gut flora-deficiency and gut glora-deficiencyApc^(min/+) mice were gavaged with 1×10⁸ colony forming units (CFU) ofS. thermophilus per day for 14 weeks, respectively. At the end of theexperiments, the whole intestinal tract of each mouse was carefullyharvested for visual examination of macroscopic tumors. It wasdiscovered that S. thermophilus-treated mice had a lower tumor number,size (FIG. 1A, FIG. 1B) compared to both the E.coli strain MG1655 andthe broth control group. Sulindac (180 ppm per 3 days) was used aspositive control.

Streptococcus Thermophilus Reduces AOM-Induced Colonic Tumorigenesis inMice

To further observe whether S.thermophilus could reduce tumorigenesis inmice, S. thermophilus was gavaged to the mice after 6 times AOMinjection for 20 weeks. The whole intestinal tract of each mouse wasexamined at the end of the experiments. Consistent results were obtainedwith Apc^(min/+) mice model (FIG. 2 ).

Streptococcus Thermophilus Inhibits the Proliferation of Colon CancerCells

To elucidate the anti-tumorigenic role of S. thermophilus on coloncells, MTT was performed to examine the cell viability. Colon cancercell lines HCT116, HT29 and Caco-2 and colon normal epithelial cell lineNCM460 were treated with S. thermophilus (multiplicity of infection(MOI)=50, 100, 200) for 4 hours. E. coli strain MG1655 and PBS were usedas control. As shown in FIG. 3 , S. thermophilus caused a significantdecrease of cell proliferation in colon cancer cell lines in a time- andMOI-dependent manner. However, for the normal epithelial cells, theinhibition effect has no significant difference.

Killed-Streptococcus Thermophilus Has No Effect

To determine whether the anti-tumor effect was cause by S. thermophilusitself or its product, live S. thermophilus were killed by autoclaving,and the killed bacteria were then exposed to colon cells. However, nodecreased viability of colon cancer cells was observed (FIG. 4 ), whichindicates that the anti-tumor effect requires living S. thermophilus.

Conditioned-medium of Streptococcus Thermophilus (St. CM) Inhibits theProliferation Of Colon Cancer Cells

To clarify the anti-tumor effect of the bacterial product, all bacteriabodies and debris in the culture supernatants were removed bycentrifugation and filtration using a 0.22-µm membrane to obtain theconditioned-medium. Colon cancer cells, namely HCT116, HT29, Caco-2, anda colon normal epithelial cell line NCM460 were co-cultured with theconditioned-medium at different dosage (12.5, 25, 50%) for 4 consecutivedays. MTT assay indicated that St.CM suppressed the proliferation ofcolon cancer cell in time- (FIG. 5 .A) and dose-dependent manner (FIG. 5.B). All these data revealed that the secreted molecules, but not thebacterial body or debris, exhibited the growth-inhibitory effect.

St. CM Promotes the Apoptosis of Colon Cancer Cells

Suppression of viability of tumor cells is usually associated withconcomitant activation of cell death pathways. The contribution ofapoptosis to the observed growth inhibition of St.CM was examined usingflow cytometry with Annexin V and propidium iodide (PI) double staining.The results showed an increase in the numbers of apoptotic cells inSt.CM-treated cells compared with BHI or Ec.CM-treated cells (FIG. 6 ).This effect was also observed in colon normal epithelial cell lineNCM460. These findings indicated that the apoptosis induced by St.CMaccounts for the anti-tumor effect in colon cancer cells.

Isolation of Tumor-Suppressive Probiotic-Secreted Molecules

To further determine the anti-tumor fraction derived from S.thermophilus, the St.CM was separated using 100-KDa molecular weightcutoff (MWCO) membranes. The cells were then exposed to 1% (vol/vol) >100-KDa fraction or < 100-KDa fraction for 24 hours, respectively.

St. CM>100-KDa Fraction Inhibits the Viability of Colon Cancer Cells

As shown in FIG. 7A, the MTT assay revealed that the St.CM>100-KDafractions exhibited a significant anti-tumor effect, which indicate theSt.CM>100-KDa molecules released from S. thermophilus inhibit theviability of colon cancer cells. Colony formation, proliferating cellnuclear antigen (PCNA) protein level and Ki-67 immunostaining in FIG.7B, FIG. 7C and FIG. 7D all revealed the same results.

St. CM>100-KDa Fraction Promotes the Apoptosis of Colon Cancer Cells

The effect of St.CM>100-KDa fraction on cell apoptosis was examinedusing flow cytometry after the treated cells were stained with Annexin Vand propidium iodide (PI). As shown in FIG. 8A, there was a significantincrease in the numbers of apoptotic cells in St.CM>100-KDafraction-treated HCT116 cells compared with the BHI>100-KDa fractiontreated group. This effect was also observed in Caco-2 cells, theproportions of apoptotic cells also significantly increased comparedwith the control group. Consistent with this finding, St.CM>100-KDafraction also caused the enhancement of protein expression ofcleaved-caspase 3, and cleaved poly (ADP-ribose) polymerase (PARP) inboth HCT116 and HT29 cell lines (FIG. 8B). These data confirmed theinhibitory effect of St.CM>100-KDa fraction on cell viability.

St. CM>100-KDa Fraction Arrests Cell Cycle at G0/G1 Phase

To determine the molecular mechanism by which St.CM>100-KDa fractionsuppresses cell proliferation, the effect of St.CM>100-KDa fraction oncell cycle distribution was investigated by flow cytometry afterpropidium iodide (PI) staining. As shown in FIG. 9 , treatment ofSt.CM>100-KDa fraction led to a decreasing tread in the number of the Sand G2-phases of HCT116 and Caco-2 cells and an increase in the G0/G1phase cells of both cell line (P<0.001). These findings indicated thatapoptosis in conjunction with cell cycle arrest, as induced bySt.CM>100-KDa fraction, accounts for the growth inhibition in coloncancer cells.

St. CM>100-KDa Fraction Inhibits Tumor Growth in Nude Mice ofSubcutaneous Xenograft model

To confirm the tumor-suppressive effect of St.CM>100-KDa fraction inCRC, it was tested whether St.CM>100-KDa fraction could suppress thegrowth of CRC cells in nude mice in vivo. A suspension of 3×10⁶ ofHCT116 cells were injected into the back of nude mice to build axenograft model. After 7 days, the St.CM>100-KDa fraction was injectedinto the tumors. The tumor sizes were measured per two days.BHI>100-KDa, BHI<100-KDa and St.CM<100-KDa fractions were used ascontrol treatment. As shown in FIG. 10 , the tumor growth of theSt.CM>100-KDa fraction-treated group was significantly slower than inthose control-treated groups. Nineteen days after injection, the micewere sacrificed, and the xenografts were excised. The tumor volume wassignificantly lower in St.CM>100-KDa fraction-treated nude mice ascompared to the control treated mice (P<0.05). The average tumor weightin the nude mice treated with St.CM>100-KDa fraction treated wassignificantly lower than that in the control treated mice (P<0.05). Theresults from the in vivo model provided further evidence of thetumor-suppressive role of St.CM>100-KDa fraction.

The Characterization of the S. Thermophilus Secreted >100-KDafraction

The anti-tumor compound in the St.CM>100-KDa fraction was furthercharacterized. Firstly, the St.CM>100-KDa fractions were digested byprotease K (PK, 50 µg/mL), the inhibition effect of the PK-inactivatedSt.CM>100-KDa fractions were assessed by MTT assay at the concentrationof 1% (vol/vol) in cell growth medium for 24 hours. As shown in FIG.11A, the inhibitory effect of St.CM>100-KDa fractions only existed inthe non-digested group. In the PK-treated group, there was nosignificant difference between the BHI>100-KDa fraction andSt.CM>100-KDa fraction, indicating that the anti-tumor molecules in theSt.CM>100-KDa fraction are proteins. To further validate this result,the St.CM>100-KDa fractions were boiled in a 100° C. water bath for 30mins to inactivate the protein in the isolated fraction. MTT assay wasperformed again, and the results were shown in FIG. 11B, indicating thesame conclusion: the anti-tumor molecules in the St.CM>100-KDa fractionare protein.

The Identification of the Probiotic Secreted Protein

To reveal what protein or proteins have the anti-tumor effect, thesecreted proteins were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (5%) (SDS-PAGE) and recycled throughin gel digestion, which was then subject to the mass spectrometry. TheHPLC spectrum indicated that the protein was β-galactosidase (FIG. 12A).To validate this result, the activity of the β-galactosidase in theprobiotic secreted >100-KDa fraction was determined. As shown in FIG.12B, the activity of β-galactosidase in St.CM>100-KDa fraction wassignificantly higher compared with the Ec.CM, BHI>100-KDa, BHI<100-KDaand St.CM<100-KDa. These results indicate that an anti-tumor fractionseparated from the St.CM contained a large amount of β-galactosidase.

The Anti-tumor Effect of S. Thermophilus Is Mediated by the Secretion ofΒ-galactosidase

To determine the functional involvement of β-galactosidase, weconstructed a mutant S. thermophilus strain (LacZ knockout) byhomologous recombination. which will cause the functionally abolish theproduction of β-galactosidase from S. thermophilus. As shown in FIG. 13, oral gavage of LacZ-knockout S. thermophilus (ST-KO) into Apc^(min/+)mice failed to protect against intestinal tumorigenesis (P> 0.05),indicating that the anti-tumor effect of S. thermophilus is mediated bythe secretion of β-galactosidase.

All patents, patent applications, and other publications, includingGenBank Accession Numbers or equivalents, cited in this application areincorporated by reference in the entirety for all purposes.

LIST OF REFERENCES

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2. Siegel RL, Miller KD, Fedewa SA, et al. Colorectal cancer statistics,2017. CA: a cancer journal for clinicians. May 06, 2017;67(3):177-193.

3. Daniel SG, Ball CL, Besselsen DG, Doetschman T, Hurwitz BL.Functional Changes in the Gut Microbiome Contribute to TransformingGrowth Factor beta-Deficient Colon Cancer. mSystems. Sep-October2017;2(5).

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5. Purcell RV, Visnovska M, Biggs PJ, Schmeier S, Frizelle FA. Distinctgut microbiome patterns associate with consensus molecular subtypes ofcolorectal cancer. Scientific reports. Sep. 14, 2017;7(1):11590.

6. Golombos DM, Ayangbesan A, O′Malley P, et al. The Role of GutMicrobiome in the Pathogenesis of Prostate Cancer: a Prospective, PilotStudy. Urology. Sep. 06, 2017.

7. Vogtmann E, Hua X, Zeller G, et al. Colorectal Cancer and the HumanGut Microbiome: Reproducibility with Whole-Genome Shotgun Sequencing.PloS one. 2016;11(5):e0155362.

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1-10. (canceled)
 11. A kit for treating cancer or reducing risk of cancer in a subject, comprising (1) a first container containing a first composition comprising an effective amount of a β-galactosidase, a culture of a β-galactosidase-producing bacterium, or an extract of the culture comprising a β-galactosidase; and (2) a second container containing a second composition comprising an effective amount of an anti-cancer therapeutic agent.
 12. The kit of claim 11, wherein the β-galactosidase-producing bacterium is S. thermophilus.
 13. The kit of claim 11, wherein the subject has a family history of cancer but has not been diagnosed with cancer.
 14. The kit of claim 11, wherein the subject has been diagnosed with cancer.
 15. The kit of claim 11, wherein the cancer is colon cancer.
 16. The kit of claim 11, wherein the first composition is a S. thermophilus culture, an extract of a S. thermophilus culture essentially free of S. thermophilus, an extract of a S. thermophilus culture essentially free of S. thermophilus and retained on a membrane with a molecular weight cut-off (MWCO) of 100 kDa following filtration, or an extract of a S. thermophilus culture comprising β-galactosidase.
 17. The kit of claim 11, wherein the first composition is formulated for oral administration.
 18. A composition for treating cancer or reducing risk of cancer comprising (1) an effective amount of a β-galactosidase, a culture of a β-galactosidase-producing bacterium, or an extract of the culture comprising a β-galactosidase; and (2) an effective amount of an anti-cancer therapeutic agent; and (3) a physiologically acceptable excipient.
 19. The composition of claim 18, wherein the β-galactosidase-producing bacterium is S. thermophilus.
 20. The composition of claim 18, wherein the cancer is colon cancer.
 21. The composition of claim 18, which is formulated for oral administration.
 22. The composition of claim 18, wherein the extract is an extract of a S. thermophilus culture essentially free of S. thermophilus, an extract of a S. thermophilus culture essentially free of S. thermophilus and retained on a membrane with a molecular weight cut-off (MWCO) of 100 kDa following filtration, or an extract of a S. thermophilus culture comprising β-galactosidase. 23-27. (canceled) 